Agglutination based processes have been used in qualitative and quantitative assays for a wide variety of bacteria, cell-surface antigens, and serum proteins, as well as several other analytes of interest. An example of a well known agglutination reaction is the reaction of bivalent antibodies with multivalent antigens to produce aggregates which can be detected and/or measured in a variety of ways. Compounds having multiepitopic receptors are capable of agglutinating particles coated with antigens or antibodies to produce agglomerates. These agglomerates can subsequently be detected using light scattering measurements.
In order to produce large, crosslinked aggregates in the agglutination reaction between bivalent antibodies and antigens, the antigens should have at least two or more specific binding sites. When the detection of monovalent haptens is desired, the reaction scheme can be modified by first preparing a multivalent form of the hapten, such as a hapten-protein conjugate. Consequently, any hapten present in a sample must compete with its multivalent form for the available specific binding sites of the antibody, resulting in a corresponding reduction of the measured amount of agglutination. Known methods for the preparation of multivalent forms of haptens include methods used to bind haptens to carriers in the preparation of immunogens.
The use of particles as carriers for the analyte, rather than the use of soluble proteins or protein conjugates as carriers, for enhanced sensitivity of visual or instrumental detection and/or measurement of agglutination or its inhibition is known. For example, a typical method used to attach particles to antibodies for use as particle reagents is adsorption of the antibodies onto the surface of suitable adsorbents. Polystyrene-based latex particles have been used extensively for this purpose.
Alternatively, particle reagents can be prepared by covalent attachment of the analyte to the surface of the particles. Polystyrene polymers have been modified to include functional groups capable of covalent protein attachment. U.S. Pat. No. 4,064,080 issued Dec. 20, 1977 discloses proteins covalently attached to styrene polymers via terminal amino phenyl groups. U.S. Pat. No. 4,181,636, issued Jan. 1, 1980, discloses carboxylated latex polymers coupled to immunologically active materials through a water soluble activating agent and their use as diagnostic reagents in agglutination tests. U.S. Pat. No. 4,210,723, issued Jul. 1, 1980, describes shell-core latex polymer particles of 0.15-1.5 .mu.m diameter having epoxy groups on the surface of the particles and the coupling of proteins through these epoxy groups. U.S. Pat. Nos. 4,401,765, issued Aug. 30, 1983 and 4,480,041, issued Oct. 30, 1984 disclose covalently bonded high refractive index particle reagents and their use in light scattering immunoassays. U.S. Pat. No. 4,703,018, issued Oct. 27, 1987, discloses high refractive index haloalkyl-functional shell-core polymers and their use in immunoassays where the shell-core polymers are covalently bonded to compounds of biological interest.
Agglutination assays are typically conducted in an inhibition format for the detection of antigens and haptens suspected to be present in liquid samples. Usually, the binding of a multivalent antibody to highly refractive particles coated with a specific binding antigen or hapten is inhibited in a competitive fashion by the antigen or hapten in the test sample (see for example, U.S. Pat. No. 4,401,765, issued Aug. 30, 1983). However, these assays are generally limited in sensitivity to antigens and haptens in the concentration range of 10.sup.-7 to 10.sup.-10 molar. Thus, while particle reagents composed of particle carriers and attached proteins provide a means for agglutination-based assays, assays such as latex particle carrier based assays often do not provide the sensitivity required for analytes present at sub-nanomolar concentrations. A continuing problem in agglutination-based immunoassays is the inability to detect analytes at concentrations below 10.sup.-10 molar.
Australian Patent Application No. 37852/89, published Jan. 11, 1990, discloses a method for the detection of a specific binding substance (SBS) which includes incubating the specific binding substance with at least three receptors; R1 and R2 which can bind to each other and R3 which specifically binds SBS, and measuring the resulting agglutination. R1 is a conjugate of one component of a specific binding pair (P) with a substance which is SBS or its analog and having at least one epitope in common; R2 has at least two binding sites for P and R3 has at least two binding sites, at least one of which binds specifically to an epitope of the SBS. Australian Patent Application No. 37852/89 does not disclose the use of a multivalent ligand having at least two binding sites which are capable of specifically binding with the same binding site on an agglutinating agent.